Thrombocytopenia, or low platelet count, is a life threatening condition associated with cytotoxic cancer treatments and other disorders. Platelet transfusion is the only current effective treatment for severe thrombocytopenia. Although effective, transfusion is associated with immunohematological side effects and infections. The aim of this project is to better understand the molecular regulation of platelet production with the ultimate goal of discovering novel targets for the development of new therapeutics for thrombocytopenia. We propose that forward genetic screens using random mutagenesis provide an unbiased, genome-wide discovery tool for regulators of circulating platelet numbers that do not rely on pre-existing knowledge of potential gene function and thus have the capacity to discover hitherto unanticipated modulators of platelet numbers. Moreover, mutagenesis screens using a murine model of thrombocytopenia will uncover genes in which loss-of function mutations lead to amelioration of disease. Since drugs overwhelmingly inhibit their targets, the proteins normally encoded by these suppressors of thrombocytopenia should provide biologically validated targets for therapeutic discovery. In preliminary studies, we have isolated several suppressors of thrombocytopenia (PIt mutants) including three that have mutations in the c-Myb transcription factor (Plt3 and Plt4) or the p300 transcriptional co-regulator/c-Myb partner protein (Plt6). Mice heterozygous for these mutations display amelioration of the thrombocytopenia inherent in mice lacking c-MpI, the receptor for thrombopoietin (TPO) and homozygotes exhibit supra-physiological platelet counts characterized by excessive megakaryocytopoiesis. We hypothesize that c-Myb/p300 activity is normally required to keep megakaryocytopoiesis in appropriate check and that pharmacological disruption of c-Myb/p300 may provide a novel therapeutic strategy to treat thrombocytopenia. The aims of this application are to fully define the biological consequences of c-Myb/p300 mutation in Plt3, Plt4 and Plt6 mice, to determine the effects of these mutations on the transcriptional regulatory functions of c-Myb/p300, to define the key target genes of this complex that control megakaryocyte and platelet production and to begin to test the potential of inhibiting c- Myb/p300 clinically by studying the responses of mice bearing c-Myb/p300 mutations in animal models of chemotherapy or bone marrow transplant-induced thrombocytopenia.